干扰MSTN对绵羊成肌细胞增殖分化及相关基因表达的影响

doi:10.11843/j.issn.0366-6964.2018.01.006

摘要/Abstract

摘要：

旨在探讨MSTN基因对绵羊成肌细胞增殖和分化的作用及相关机制，进一步揭示MSTN在绵羊成肌细胞中的生物学功能。本研究利用重组腺病毒介导shRNA干扰绵羊成肌细胞内源性MSTN基因表达，通过CCK-8法检测成肌细胞增殖能力，流式细胞仪检测细胞周期变化，qRT-PCR检测p21基因表达。成肌细胞诱导分化后利用免疫细胞化学技术检测肌管的形成情况，统计细胞融合率，qRT-PCR检测诱导48、72、96 h后 MyoD、MyoG、Myf5、Myf6和HACD1基因的表达量变化。结果发现，干扰MSTN后成肌细胞的增殖受到抑制，细胞周期停滞在G0/G1期，并且p21的表达呈上升趋势。在对成肌细胞分化作用的研究中，发现干扰组与阴性对照组相比，诱导48 h 4个生肌调节因子的表达量均呈下降趋势，MyoG基因表达显著下调（P<0.05）；诱导72 h干扰组成肌细胞融合率极显著高于阴性对照组（P<0.01），Myf5和Myf6基因表达量极显著降低（P<0.01），MyoD基因表达量升高不显著（P>0.05），MyoG基因表达量极显著增加（P<0.01），诱导96 h，Myf5基因表达量显著降低（P<0.05），MyoD基因表达量降低不显著（P>0.05），MyoG基因表达量极显著降低（P<0.01），Myf6基因表达量升高不显著（P>0.05）。另外诱导72 h，HACD1基因的表达量在干扰组中显著高于阴性对照组（P<0.05），诱导48和96 h无显著变化。研究表明，干扰MSTN表达对成肌细胞的增殖有抑制作用，对分化有促进作用，HACD1基因与成肌细胞融合相关。

Abstract:

The purpose of this study was to explore the regulation mechanism of MSTN gene on the proliferation and differentiation of sheep myoblasts and further reveal the biological function of MSTN. The expression of endogenous MSTN gene of sheep myoblasts was interfered by recombinant adenovirus-mediated shRNA. CCK-8 assay was used to detect the proliferation of myoblasts, and flow cytometry analysis was used to detect cell cycle of myoblasts. The qRT-PCR was used to measure the mRNA expression of p21 in myoblasts. Immunocytochemistry was used to detect myotubes formation and the cell fusion rate was counted, qRT-PCR was used to measure the mRNA expression of the MyoD,MyoG,Myf5, Myf6 and HACD1 genes at 48, 72, 96 h after myoblast induced to differentiation. The results showed that the proliferation of myoblasts was inhibited after downregulating MSTN expression, the cell cycle was arrested at G0/G1 phase, and the expression of cell cycle inhibitor p21 increased. In the study on myoblast differentiation, we found the expression of 4 myogenic regulatory factors after inducing for 48 h were decreased, and the expression of MyoG gene decreased significantly(P<0.05);the myoblast fusion rate of interference group was significantly higher than that of negative control group(P<0.01) after inducing for 72 h, the expression of Myf5 and Myf6 genes decreased significantly (P<0.01), the expression of MyoD gene didn't increase significantly (P>0.05), the expression of MyoG gene increased significantly(P<0.01);the expression of Myf5(P<0.05), MyoD(P>0.05) and MyoG(P<0.01) decreased after inducing for 96 h, and the expression of Myf6 gene didn't increase significantly(P>0.05). In addition, the expression of HACD1 gene was significantly higher in the interference group than that in the negative control group (P<0.05) after inducing for 72 h, and no significant changes were detected after inducing for 48 or 96 h. The results show that the MSTN gene interfered could inhibit the proliferation while promote differentiation of myoblasts, and HACD1 gene is related to myoblast fusion.

中图分类号:

S826.2

王红娜, 孙洪新, 张英杰, 刘月琴, 谷振慧, 史秀芬. 干扰MSTN对绵羊成肌细胞增殖分化及相关基因表达的影响[J]. 畜牧兽医学报, 2018, 49(1): 46-54.

WANG Hong-na, SUN Hong-xin, ZHANG Ying-jie, LIU Yue-qin, GU Zhen-hui, SHI Xiu-fen. Effects of Interfering MSTN on Proliferation and Differentiation of Sheep Myoblasts and Expression of Related Genes[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(1): 46-54.

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